The field of the invention is molecular markers for muscle stem cells.
The development, growth, and repair of skeletal muscles all require mononucleate myoblasts that are committed to form multinucleate myofibers via intercellular fusion. Committed myoblasts are thought to be the progeny of uncommitted, self-renewing stem cells; however, molecular markers that would permit identification and study of muscle stem cells have not previously been described (Quinn et al., Exp. Cell Res. 154, 65-82 (1984); Baroffio et al., Differentiation 60, 47-57 (1996)). These and all other publications and patents cited herein are hereby incorporated by reference.
It has now been discovered that Bcl-2, an apoptosis-inhibiting protein, is expressed in muscle stem cells, but not in other myogenic cells (e.g., multinucleate myotubes and myofibers). Thus, Bcl-2 is a molecular marker for muscle stem cells (e.g., human muscle stem cells). This discovery suggests a number of methods for identifying and/or isolating muscle stem cells. For example, one method of the invention provides a means for identifying a muscle stem cell by providing a sample that includes a myogenic cell and detecting activity of a Bcl-2 promoter within the myogenic cell as an indication that the myogenic cell is a muscle stem cell. The invention can be used to detect muscle stem cells that produce skeletal muscle, smooth muscle, or cardiac muscle. The Bcl-2 marker also can be used to detect and characterize a stem cell component in muscle tumors (e.g., in methods of diagnosing or evaluating muscle tumors).
The activity of the Bcl-2 promoter can be detected by any of a variety of methods. For example, the activity of the Bcl-2 promoter can be detected by detecting a Bcl-2 protein in the myogenic cell. To this end, conventional methods, such as SDS-PAGE and/or immunoassays, can be employed. Antibodies that specifically bind Bcl-2 are known in the art and readily available for use in such immunoassays. If desired, the activity of the Bcl-2 promoter can be detected by detecting Bcl-2 mRNA in the myogenic cell. Art-known methods such as reverse transcription-PCR (RT-PCR), in situ hybridization, and Northern blots can be used to detect the Bcl-2 mRNA.
In a variation of the above methods, the activity of a Bcl-2 promoter is detected with the use of a heterologous reporter gene (e.g., a chloramphenicol acetyltransferase gene, an alkaline phosphatase gene, a luciferase gene, or a green fluorescent protein gene). In a typical method, the heterologous reporter gene is operably linked to a Bcl-2 promoter in a genetic construct (e.g., a viral-based vector or a plasmid). Conventional molecular biology techniques can be used to produce such a genetic construct. The genetic construct then is introduced into a population of cells containing myogenic cells and thought to contain muscle stem cells. Since no myogenic cells except muscle stem cells activate the Bcl-2 promoter, expression of the reporter gene is detected as an indication that a cell is a muscle stem cell. As above, conventional methods for detecting gene expression can be used to detect reporter gene expression (e.g., protein or mRNA assays).
Now that a molecular marker for identifying muscle stem cells has been discovered, several related methods are possible. Thus, the invention also provides a method for determining whether a test compound(s) modulates muscle stem cell differentiation. In this method, a cell is identified as a muscle stem cell (e.g., by using one of the abovedescribed methods). The muscle stem cell is contacted with the test compound (e.g., in vitro). A change in the differentiation of the stem cell, compared to control, is an indication that the compound modulates muscle stem cell differentiation. Any compound can be used as the test compound in this method. Both naturally-occurring and synthetic polypeptides and small organic molecules are suitable test compounds. Compounds and analogs of compounds that are known to affect the differentiation of other cells are particularly suitable for use in this method. Parameters such as the rate and pattern of cell differentiation can be measured using conventional means. In a related method, one can determine whether a test compound modulates muscle stem cell proliferation. This method is nearly identical to that described above, except that a change in cell proliferation, compared to control, is detected. Of particular interest are test compounds that modulate the rate of cell proliferation. Of course, the above-described methods for detecting compounds that modulate cell differentiation and proliferation can be combined into a single experiment using one or more test compounds.
The invention also provides a method for producing a population of cells that is enriched for muscle stem cells relative to a reference population of cells. The method entails providing a reference population of cells that includes a plurality of muscle stem cells and at least one cell (typically many cells) other than a muscle stem cell (e.g., myoblasts). Typically, the reference population is obtained by muscle biopsy. A genetic construct may be introduced into the reference population of cells. The genetic construct includes a Bcl-2 promoter that is operably linked to a gene encoding a marker protein. The marker protein is a protein that is heterologous to wild-type cells of the reference population. Cells that express the marker protein (i.e., cells in which the Bcl-2 promoter is active) are then isolated in order to produce a population of cells enriched for muscle stem cells. Of course, by removing the Bcl-2-expressing cells from the cell population, this method can be used to produce a population of cells depleted of muscle stem cells.
The heterologous marker protein can be viral, prokaryotic, eukaryotic, or synthetic in origin. Preferably, the marker protein is not naturally expressed in wild-type muscle stem cells or muscle cells in general. Typically, the marker protein is a polypeptide that is expressed on the cell surface. Examples of suitable marker proteins include CD8, xcex2-galactosidase, green fluorescent protein, catechol 2,3-dioxygenase, aequorin, and influenza virus hemagglutinin (which can be detected using commercially available monoclonal antibodies); the genes encoding these and other suitable marker proteins are known in the art. Conventional cell sorting methods (e.g., fluorescence-activated cell sorting (FACS)) can be used to isolate those cells in which the Bcl-2 promoter directs the expression of the gene encoding the marker protein. Other techniques, such as the use of protein-conjugated magnetic beads that selectively bind particular cells, also can be used. For example, magnetic beads conjugated to anti-CD8 antibodies can be used to isolate muscle stem cells expressing CD8 under the control of the Bcl-2 promoter.
Included within the invention is a method for producing a population of living cells enriched for muscle stem cells relative to a reference population of cells (i.e., a starting population of cells). This method entails:
(a) providing a reference population of living cells that includes a plurality of muscle stem cells that express Bcl-2 and at least one cell other than a muscle stem cell (e.g., a myoblast); and
(b) treating the reference population of cells to induce apoptosis (i.e., programmed cell death) in cells that do not express Bcl-2, thereby producing a population of living cells enriched for muscle stem cells. The expression of Bcl-2 inhibits apoptosis of the muscle stem cells, thereby allowing the muscle stem cells to survive under conditions that result in the death of other cells. In this method, apoptosis can be induced by any of the art-known methods. In a preferred method, the cells are contacted with staurosporine (C28H26N4O3) in a serum-free cell culture medium. Of course, the surviving muscle stem cells can then be separated from the non-living cells in the cell sample.
The discovery of a molecular marker for muscle stem cells makes it now possible to express an exogenous coding sequence in a muscle stem cell specifically. Thus, the invention also includes a method of expressing an exogenous coding sequence in a muscle stem cell; this method entails:
(a) identifying a myogenic cell as a muscle stem cell;
(b) introducing into the muscle stem cell a genetic construct comprising an exogenous coding sequence operably linked to a muscle stem cell-active promoter, to produce a transfected muscle stem cell; and
(c) maintaining the transfected muscle stem cell containing the genetic construct under conditions permitting expression of the exogenous coding sequence. The above-described methods for identifying and/or isolating muscle stem cells by detecting or exploiting the activity of a Bcl-2 promoter can be used in this aspect of the invention.
Preferably, the genetic construct includes a viral vector (i.e., all or a portion of a viral genome). In addition, the genetic construct typically contains a promoter that is active in muscle stem cells (e.g., a Bcl-2 promoter) and which is operably linked to the exogenous gene.
The genetic construct can be introduced into the muscle stem cell in vitro or in vivo. If desired, once a genetic construct has been introduced into a muscle stem cell, the cell subsequently can be introduced into a mammal (e.g., a human or mouse) and maintained under conditions such that the exogenous coding sequence is expressed in the mammal.
The term xe2x80x9cBcl-2xe2x80x9d is used herein in accordance with its ordinary definition in the art. The Bcl-2 protein is considered to be an apoptosis-inhibiting, membrane-associated cytoplasmic protein having a molecular weight approximately 26 kD (Tsujimoto et al., 1987, Oncogene 2:3; see also U.S. Pat. Nos. 5,202,429 and 5,015,568). A nucleotide sequence encoding Bcl-2 has been described (Tsujimoto and Croce, 1986, Proc. Natl. Acad. Sci. 83:5214-5218 and GenBank Accession number M13994 under the locus identification HUMBCL2A). A further description of Bcl-2 is provided by Korsmeyer (1995, Trends in Genet. 11:101-105). Preferably, the Bcl-2 protein is a human protein, although Bcl-2 proteins from other species (e.g., mice) also can be used.
By xe2x80x9cmuscle stem cellxe2x80x9d is meant a self-renewing mononucleate cell that produces as progeny mononucleate myoblasts, which are committed to form multinucleate myofibers via intercellular fusion. Encompassed by the invention are muscle stem cells that produce skeletal muscle, smooth muscle, or cardiac muscle.
xe2x80x9cMyogenicxe2x80x9d cells as described herein are those cells that are related to the origin of muscle cells or fibers. Various molecular markers are known to be specific for the middle and late stages of myogenic differentiation. For example, in C2C12 cells, myosin and MRF4 mark the late stages of myogenesis and are largely restricted to myotubes, whereas myogenin and nestin mark the middle stages of myogenesis and are found in all myotubes and in many committed myoblasts.
By xe2x80x9cpromoterxe2x80x9d is meant a minimal nucleotide sequence sufficient to direct transcription of a coding sequence. Included within the invention are those promoters which are inducible by external signals or agents; such elements can be located in the 5xe2x80x2 or 3xe2x80x2 untranslated regions of the native gene. A xe2x80x9cBcl-2 promoterxe2x80x9d is any sequence contained within the untranslated region of the endogenous Bcl-2 gene that is sufficient to direct transcription of Bcl-2 in muscle stem cells, and which does not direct expression of Bcl-2 in myoblasts or myotubes. For example, a 1.8 kb sequence immediately adjacent to the Bcl-2 transcription start site is sufficient to direct gene expression in muscle stem cells but not myoblasts or myotubes. It is recognized that, in producing genetic constructs containing a Bcl-2 promoter (e.g., those constructs that also contain a reporter gene or a gene encoding a marker protein), minor variations (e.g., deletions, point mutations, and the like) can be made in the sequence of the Bcl-2 promoter without abrogating its ability to be active in muscle stem cells and inactive in other myogenic cells. Thus, Bcl-2 promoters having such minor variations without abrogating the muscle stem cell specificity of the promoter are encompassed by the term xe2x80x9cBcl-2 promoter.xe2x80x9d In addition, multiple copies of the Bcl-2 promoter, arranged in tandem, can be used to direct gene expression.
By xe2x80x9coperably linkedxe2x80x9d is meant that a coding sequence and a regulatory sequence(s) (e.g., a promoter) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s).
The term xe2x80x9cexogenousxe2x80x9d refers to any coding sequence, promoter, polypeptide or other molecule that is supplied to the muscle stem cell (e.g., as part of a genetic construct). Included are those coding sequences that normally are present in the muscle stem cell as well as coding sequences that are not normally present in the muscle stem cell into which the genetic construct is introduced (e.g., related and unrelated genes of other cells or species).
The term xe2x80x9cheterologousxe2x80x9d refers to any gene, promoter, polypeptide or other molecule that is not naturally present in a wild-type version of a referenced cell. For example, an E. coli xcex2-galactosidase gene is considered to be xe2x80x9cheterologousxe2x80x9d to a human muscle stem cell.
The term xe2x80x9creporter genexe2x80x9d refers to any gene for which gene expression can be monitored. Commonly used reporter genes include, for example, genes encoding chloramphenicol acetyltransferase, alkaline phosphatase, luciferase, and green fluorescent protein.
By xe2x80x9cdifferentiationxe2x80x9d is meant the developmental process whereby cells become specialized, i.e., acquire one or more characteristics or functions different from that of the original cell type.
By xe2x80x9cproliferationxe2x80x9d is meant an increase in number of cells.
By xe2x80x9cmarker proteinxe2x80x9d is meant a polypeptide that distinguishes one cell (or set of cells) from another cell (or set of cells) in a population of cells. For example, a polypeptide that is expressed (e.g., by genetic engineering) on the surface of muscle stem cells but not other cells of a cell population serves as a marker protein for the muscle stem cells. Typically, the marker protein is a cell-surface antigen, such that antibodies that specifically bind the marker protein can be used in cell sorting methods, e.g., to produce a population of cells enriched for cells that express the marker protein. Alternatively, intracellular proteins can be used as marker proteins. For example, fluorescent or luminescent proteins, such as green fluorescent protein and aequorin of Aequoria victoria (Tanahashi et al., Gene 96:249-255 (1990)) can be used as the marker protein and can facilitate cell sorting, e.g., by FACS. Also, enzymes can be used, provided that the activity of the enzyme can be detected. For example, xcex2-galactosidase is well suited for use as a marker protein; this enzyme can be detected by introducing into the cell a substrate(s) that releases a fluorescent product(s) upon cleavage by the enzyme (available from, e.g., Molecular Probes). Another suitable enzyme is catechol 2,3-dioxygenase, which is encoded by xylE of Pseudomonas putida (Domen et al., Analy. Biochem. 155:379-384 (1986)).
By xe2x80x9capoptosisxe2x80x9d is meant the physiological process known as programmed cell death. Unlike other forms of cell death that occur, apoptosis is an active, ATP-requiring form of cell death that typically requires new RNA and protein synthesis. Generally, apoptosis is characterized by the activation of endogenous endonucleases that degrade genomic DNA.
The invention offers the advantage of providing a convenient molecular marker for muscle stem cells. Now that such a marker has been identified, muscle stem cells can readily be isolated from, and/or characterized in, a mixed population of cells. Also, muscle stem cells, as distinct from myoblasts and myofibers, now can be used selectively to express an exogenous gene. These muscle stem cells are expected to be more effective in gene therapy methods than other muscle cells.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.